Spin Columns Efficiency

Cathal Garvey via methods%40net.bio.net (by cathalgarvey from gmail.com)
Fri Feb 5 03:44:32 EST 2010


Also important is the gel buffer used: avoid TBE for gel extractions, as
Boric acid at that molarity can bind DNA and inhibit downstream applications
such as spin columns.

I would recommend looking up Sodium Borate as a gel buffer. It permits
higher voltages without heating, which saves time, it permits downstream
applications and gel extractions, and it offers good resolution. Also, it's
about 35 times cheaper.

If you want to try it out, make up 1x Sodium Borate buffer with 1.907g of
disodium Borate decahydrate in 1l of distilled water, optionally filter
sterilise.

Also see the faq on fasterbettermedia.com.

All the best,
Cathal

On Feb 5, 2010 5:31 AM, "Adam ." <anonwums1 from gmail.com> wrote:

The binding efficiency of spin columns depends on pH and salt concentration.
Generally the binding buffer you dissolve the gel slice in should have
sufficient salt and pH. The columns work really badly to bind DNA if the pH
is greater than 7.5. Some protocols suggest that you spike in a little 3 M
sodium acetate pH 5 before running it on the column. Elution off of the
column is also pH and salt dependent. You want a higher pH buffer to elute
in.

There are also binding issues with the size of the DNA fragment. There is an
optimal range of size over which the columns are supposed to work.

Generally, most of this stuff is found in the back of the manual for your
spin column. You might get some insight by calling up technical support. In
my experience, even though the manufacturer claims you get 99% recovery, you
rarely do and are lucky if you get 50-70%.

Adam

On Thu, Feb 4, 2010 at 7:41 PM, Adroit <cognizann from googlemail.com> wrote: >
Dear Bionetters, > > > ...


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